Project Summary The long-term objective of this work is to understand the biochemical mechanism of the telomerase enzyme. Telomerase is a highly specialized reverse transcriptase (RT), responsible for the maintenance of the telomeric ends capping linear eukaryotic chromosomes. This is wholly essential for genome stability and central to human cellular aging, cancer biology and disease. Telomerase synthesizes telomeric DNA repeats by reverse transcribing a precisely defined short template within the vastly larger internal telomerase RNA (TR) component. For human telomerase, we have recently reported that the TR template self-defines its boundary through an exceptionally unusual mechanism, whereby telomerase recognizes a single-nucleotide embedded in the DNA product to terminate DNA synthesis at the end of the template. We will build on this exciting discovery by investigating the mechanism for which this pause signal is recognized and facilities the complex telomerase catalytic cycle. Beyond merely providing the RNA template for DNA synthesis, the TR component contains essential and evolutionarily conserved RNA structural domains: the pseudoknot and CR4/5. The catalytic telomerase RT (TERT) protein component contains the hallmark RT motifs that constitute the catalytic site for DNA synthesis. However, the TERT protein is absolutely reliant on pseudoknot and CR4/5 association for catalytic activity. Disease mutations that damage these critical RNA domains within TR abolish TERT protein activity. The mechanism underlying TERT reliance on TR for functionality has remained elusive until our recent breakthrough with the mapping the TERT-CR4/5 binding interface has directly lead to the testable hypothesis of CR4/5 acting as an allosteric regulator. We are now well positioned to interrogate the mechanism for CR4/5, and map the TERT-pseudoknot binding interface, to reveal their precise functionality. This research project focuses on illuminating the molecular mechanisms of telomerase function, encompassing both the unique telomerase-specific phenomenon of sequence-dependent pausing and the absolute reliance on RNA components for TERT protein catalysis. We will employ state-of-the-art systems and innovative assays that we have pioneered and established for dissecting specific telomerase catalytic steps, mapping TERT-TR binding interface(s) and articulating detailed TR domain functionality. The highly specialized and unique function of the telomerase enzyme requires high-level synergy among the myriad of domains comprising the TERT and TR components. Successful outcomes of these aims will provide much- needed details necessary for understanding the complex inner workings of telomerase action.